The present invention relates to genetically-modified non-human mammals and genetically-modified animal cells containing a functionally disrupted PDE11A gene. The invention also features methods of screening for agents that modulate PDE11A and methods of modulating cAMP and cGMP signal transduction in cells that express PDE11A.
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic nucleotides, such as the second messengers cAMP (cyclic adenosine 3xe2x80x25xe2x80x2-monophosphate) and cGMP (cyclic guanine 3xe2x80x25xe2x80x2-monophosphate). Thus, PDEs play a pivotal regulatory role in a wide variety of signal transduction pathways (Beavo, Physiol. Rev. 75: 725-48, 1995). For example, PDEs mediate processes involved in vision (McLaughlin et al., Nat. Genet. 4: 130-34, 1993), olfaction (Yan et al., Proc. Natl. Acad. Sci. USA 92: 9677-81, 1995), platelet aggregation (Dickinson et al. Biochem. J. 323: 371-77, 1997), aldosterone synthesis (MacFarland et al., J. Biol. Chem. 266: 136-42, 1991), insulin secretion (Zhao et al., J. Clin. Invest. 102: 869-73, 1998), T cell activation (Li et al., Science 283: 848-51, 1999), and smooth muscle relaxation (Boolell et al., Int. J. Impot. Res. 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-71, 1998).
PDEs form a superfamily of enzymes that are subdivided into 11 major families (Beavo, Physiol. Rev. 75: 725-48, 1995; Beavo et al., Mol. Pharmacol. 46: 399-05, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-96, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-77, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: 751-56, 1998; Soderling et al., J. Biol. Chem. 273: 15553-58, 1998; Fisher et al., J. Biol. Chem. 273: 15559-64, 1998; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-76, 1999; and Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000).
Each PDE family is distinguished functionally by unique enzymatic characteristics and pharmacological profiles. In addition, each family exhibits distinct tissue, cell, and subcellular expression patterns (Beavo et al., Mol. Pharmacol. 46: 399-405, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-96, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-77, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: 751-56, 1998; Soderling et al., J. Biol. Chem. 273: 15553-58, 1998; Fisher et al., J. Biol. Chem. 273: 15559-64, 1998; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-76, 1999; Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000; Boolell et al., Int. J. Impot. Res. 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-71, 1998; Houslay, Semin. Cell Dev. Biol. 9: 161-67, 1998; and Torphy et al., Pulm. Pharmacol. Ther. 12: 131-35, 1999). Therefore, by administering a compound that selectively regulates the activity of one family or subfamily of PDE enzymes, it is possible to regulate cAMP and/or cGMP signal transduction pathways in a cell- or tissue-specific manner.
PDE11 is one of the most recently described families of PDEs; PDE11A is the sole member of this family so far identified (Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000, hereinafter xe2x80x9cFawcett, 2000,xe2x80x9d Yuasa et al., J. Biol. Chem. 275: 31469-79, 2000, hereinafter xe2x80x9cYuasa, 2000xe2x80x9d). While PDE11A is known to be expressed in, e.g., testis, skeletal muscle, kidney, liver, various glandular tissue (e.g., pituitary, salivary, adrenal, mammary, and thyroid), pancreas, spinal cord, and trachea (Fawcett, 2000), little is known about PDE11A function. The present invention provides biological tools to study PDE11A function and methods to identify agents that regulate PDE11A activity for use in treating diseases and conditions that are linked to these PDE11A functions.
One embodiment of the present invention is the provision of a PDE11A knockout mouse, which provides an excellent opportunity to investigate genes involved in, inter alia, spermatogenesis, and, when compared with the wildtype mouse, to dissect out the components involved in spermatogenesis. One method for this type of analysis is microarray technology. With DNA microarray technology, it becomes possible to monitor large-scale gene expression over time. Prefabricated arrays of large numbers of especially designed oligonucleotide probes, e.g. as manufactured by Affymetrix (CA, USA), enable simultaneous hybridization-based analysis of thousands of genes.
The present invention features genetically-modified animal cells and genetically-modified non-human mammals containing a disrupted PDE11A gene, as well as assays for identifying PDE11A function in cells and tissues that express PDE11A, methods for identifying agents that modulate these PDE11A functions, and methods of treating or preventing diseases or conditions in mammals by modulating PDE11A function. For example, modulators of PDE11A activity are administered to a mammal to modulate spermatogenesis.
One aspect of the invention features a genetically-modified, non-human mammal, wherein the modification results in a functionally disrupted PDE11A gene. Preferably, the mammal is heterozygous for the modification. More preferably, the mammal is homozygous for the modification. In other preferred embodiments, the mammal is a rodent, more preferably, a mouse.
In a second aspect, the invention provides a genetically-modified animal cell, wherein the modification comprises a functionally disrupted PDE11A gene. Preferably, the animal cell is heterozygous for the modification. More preferably, the cell is homozygous for the modification. In other preferred embodiments, the cell is an embryonic stem (ES) cell, and/or the cell is human or murine.
The third aspect of the invention features a method of identifying an agent that modulates spermatogenesis, involving contacting an agent with a PDE11A polypeptide and measuring PDE11A activity, wherein a difference between the activity in the absence of the agent and in the presence of the agent is indicative that the agent can modulate spermatogenesis. Preferably, the method identifies an agent that inhibits PDE11A activity for use in decreasing spermatogenesis.
In a related fourth aspect, the invention also provides a method of identifying an agent that modulates spermatogenesis, which includes contacting an agent with a cell that expresses a PDE11A polypeptide and measuring PDE11A activity or PDE11A expression, wherein a difference between the activity or expression in the absence of the agent and in the presence of the agent is indicative that the agent can modulate spermatogenesis.
The fifth aspect provides a method of modulating spermatogenesis in a mammal, the method comprising administering an agent that modulates PDE11A activity. Preferably, the agent administered reduces PDE11A activity and reduces spermatogenesis.
Featured in the sixth aspect is a method of modulating cAMP and/or cGMP-mediated signal transduction in a mammal in testis, prostate, pituitary gland, bladder urothelium and/or bladder nerve fibers, neurons, skeletal muscle, cardiac myocytes, vascular smooth muscle, and/or vascular endothelial cells, which includes administering an agent that modulates PDE11A activity. In preferred embodiments, the agent selective for PDE11A, or is UK-336,017 (IC-351), UK-227,786 (E4021), or UK-235,187.
The invention also features in a seventh aspect, a method of treating hypertension, cardiac insufficiency, atherosclerosis, hyperprolactinemia, growth hormone insufficiency, incontinence, or disorders associated with skeletal muscle metabolism or contractility, wherein the method comprises administering an agent that modulates PDE11A activity. Preferably, the agent is selective for PDE11A. More preferably, the agent is UK-336,017 (IC-351), UK-227,786 (E4021), or UK-235,187.
In an eighth aspect, the invention provides biomarkers indicative of modulated spermatogenesis in a mammal; these biomarkers can be used to monitor spermatogenesis in a mammal as well as for monitoring diseases or disorders characterised by reduced spermatogenesis. These biomarkers are useful in the clinical assessment of spermatogenesis, and can be used to aid in the selection of the appropriate therapeutic intervention. The biomarkers of the present invention are selected from the group consisting of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 and Krox-24 binding protein.
In a related ninth aspect, the invention provides biomarkers indicative of modulated cAMP and/or cGMP-mediated signal transduction in a mammal in testis, prostate, pituitary gland, bladder urothelium and/or bladder nerve fibers, neurons, skeletal muscle, cardiac myocytes, vascular smooth muscle, and/or vascular endothelial cells.
In a tenth aspect of the present invention, there is provided a method of modulating spermatogenesis in a mammal, said method comprising administering a modulator of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein.
In a related eleventh aspect, the invention provides a method of modulating cAMP and/or cGMP-mediated signal transduction in a mammal in testis, prostate, pituitary gland, bladder urothelium and/or bladder nerve fibers, neurons, skeletal muscle, cardiac myocytes, vascular smooth muscle, and/or vascular endothelial cells, said method comprising administering a modulator of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein.
Preferably, said modulator of the tenth and eleventh aspects of the invention is an inhibitor or antagonist of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein. More preferably, said inhibitor or antagonist is an antisense oligonucleotide or ribozyme against, or a compound that down-regulates the transcription and/or translation of, any gene(s) which encode(s) any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein.
In a preferred embodiment of the invention, if a male contraceptive agent is desired (i.e. to reduce spermatogenesis), then said agent will comprise an inhibitor or antagonist of any one or more Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen or HR6A. Such an agent will essentially have the effect of decreasing the presence in the testis of any one or more Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen or HR6A.
In another preferred embodiment of the invention, if a male pro-fertility agent is desired (i.e. to increase or normalize spermatogenesis), then said agent will comprise an inhibitor or antagonist of any one or more Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein. Such an agent will essentially have the effect of decreasing the presence in the testis of any one or more Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein. The term xe2x80x9cnormalizexe2x80x9d is used in the present context to denote an increase in spermatogenesis, where the starting base level of spermatogenesis is below normal levels. This compares to the use of the term xe2x80x9cincreasexe2x80x9d, which is used in the present context to denote an increase in spermatogenesis, where the starting base level of spermatogenesis is already at a normal level.
Alternatively, said modulator of the tenth and eleventh aspects of the invention is a stimulator, activator or agonist of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein. Preferably, said stimulator or activator is a stimulator or activator of the transcription and/or translation of any gene(s) which encode(s) any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein.
In a preferred embodiment of the invention, if a male contraceptive agent is desired (i.e. to reduce spermatogenesis), then said agent will comprise a stimulator, activator or agonist of any one or more Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein. Such an agent will essentially have the effect of increasing the presence in the testis of any one or more Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein.
In another preferred embodiment of the invention, if a male pro-fertility agent is desired (i.e. to increase or normalize spermatogenesis), then said agent will comprise a stimulator, activator or agonist of any one or more Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen or HR6A. Such an agent will essentially have the effect of increasing or normalizing the presence in the testis of any one or more Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen or HR6A. The term xe2x80x9cnormalizexe2x80x9d is used in the present context to denote an increase in spermatogenesis, where the starting base level of spermatogenesis is below normal levels. This compares to the use of the term xe2x80x9cincreasexe2x80x9d, which is used in the present context to denote an increase in spermatogenesis, where the starting base level of spermatogenesis is already at a normal level.
According to a twelfth aspect of the invention, there is provided use of a modulator of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein, in the manufacture of a medicament for modulating spermatogenesis in a mammal.
According to a thirteenth aspect of the invention, there is provided use of a modulator of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein, in the manufacture of a medicament for modulating cAMP and/or cGMP-mediated signal transduction in a mammal in testis, prostate, pituitary gland, bladder urothelium and/or bladder nerve fibers, neurons, skeletal muscle, cardiac myocytes, vascular smooth muscle, and/or vascular endothelial cells.
Preferably, said modulator of the twelfth and thirteenth aspects of the invention is an inhibitor or antagonist of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein. More preferably, said inhibitor or antagonist is an antisense oligonucleotide or ribozyme against, or a compound that down-regulates the transcription and/or translation of, any gene(s) which encode(s) any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein.
Alternatively, said modulator of the twelfth and thirteenth aspects of the invention is a stimulator, activator or agonist of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein. More preferably, said stimulator or activator is a stimulator or activator of the transcription and/or translation of any gene(s) which encode(s) any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein.
It will be appreciated that the inhibitors or antagonists of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein can be compounds that reduce the activity of the target, antibodies specific for the target, as well as antisense oligonucleotides or ribozymes or transcription inhibitors which have the effect of reducing the amount of the target produced. It will also be appreciated that stimulators, activators or agonists of any one or more of Corticosteroid binding globulin, Centrin 3, XRCC1, Chromobox M33, GABA-A (gamma 3 sub-unit), Prohormone convertase 5, Leydig Insulin-like peptide, Calpain 3, Y-Box 3, Chromogranin B, Cryptdin I, PP2B, Glutamate cysteine ligase, Nidogen, HR6A, Protamine 1, sp32, mCDC46, Adenylate kinase 2, AKAP121 or Krox-24 binding protein can comprise compounds that increase the activity of the target itself, as well as compounds that increase the expression of the target; another way to achieve increased activity of the target is to deliver the target itself, either as protein or its coding region in an appropriate vector as gene therapy.
According to a fourteenth aspect of the invention, there is provided use of agent that modulates PDE11A activity in the manufacture of a medicament for the modulation of spermatogenesis. Preferably, said agent reduces PDE11A activity and said modulation is reduction of spermatogenesis.
According to a fifteenth aspect of the invention, there is provided use of an agent that modulates PDE11A activity in the manufacture of a medicament for the modulation of cAMP and/or cGMP-mediated signal transduction in a mammal in testis, prostate, pituitary gland, bladder urothelium and/or bladder nerve fibers, neurons, skeletal muscle, cardiac myocytes, vascular smooth muscle, and/or vascular endothelial cells.
According to a sixteenth aspect of the invention, there is provided use of an agent that modulates PDE11A activity in the manufacture of a medicament for the treatment of hypertension, cardiac insufficiency, atherosclerosis, hyperprolactinemia, growth hormone insufficiency, incontinence, or disorders associated with skeletal muscle metabolism or contractility.
Preferably, the agent of the fourteenth, fifteenth and sixteenth aspects of the present invention is UK-336,017 (IC-351), UK-227,786 (E4021), or UK-235,187.
It will be appreciated that the present invention provides that the modulation of the PDE11A activity can be either a reduction in PDE11A activity, leading to, inter alia, a reduction in spermatogenesis, or an increase in PDE11A activity, leading to, inter alia, an increase in or normalization of spermatogenesis. Agents of the invention that result in a reduction in spermatogenesis can be classed as male contraceptive agents. Agents of the invention that result in an increase in or normalization of spermatogenesis can be classed as male pro-fertility agents.
Those skilled in the art will fully understand the terms used herein in the description and the appendant claims to describe the present invention. Nonetheless, unless otherwise provided herein, the following terms are as described immediately below.
By xe2x80x9ccoding sequencexe2x80x9d or xe2x80x9ccoding regionxe2x80x9d is meant a nucleic acid molecule having sequence information necessary to produce a gene product when the sequence is expressed.
By xe2x80x9cantisense oligonucleotidexe2x80x9d is meant a small nucleic acid molecule, typically between about 10 to 50 nucleotides long, which may be unmodified RNA or DNA or modified RNA or DNA; it is designed to hybridize to the respective transcript, and thereby reduce its translation into protein, e.g. by blocking translation or by facilitating rapid degradation of the respective transcript.
By xe2x80x9cribozymexe2x80x9d is meant a nucleic acid molecule, which can be unmodified RNA or DNA or modified RNA or DNA, which is designed to hybridize and cleave the respective transcript.
The term xe2x80x9cantibodyxe2x80x9d as used herein includes polyclonal and monoclonal antibodies, single chain, chimeric and humanized antibodies, as well as antibody fragments, whether produced by recombinant or proteolytic means. The term is also meant to include the products of any antibody-derived expression libraries, e.g. single-chain Fv or Fab fragment expression libraries.
By xe2x80x9cbiomarkerxe2x80x9d or xe2x80x9cmarkerxe2x80x9d is meant any molecule, e.g. a transcript of a gene or the translation product thereof, which is indicative of a condition, disorder, disease or dysfunction, or the status in the progression of a process, e.g. spermatogenesis, or the status in the progression of a condition, disorder, disease or dysfunction. For example, a particular gene may be down-regulated (reduced expression) in a particular disease. In such a disease, therefore, transcripts or translation products of the particular gene will be less abundant than the same transcripts or translation products in a non-diseased individual. Thus, since the abundance of particular transcripts or translation products may be ascertained using, for example, microarray technology, the particular gene (or rather its transcripts or translation products) will act as a marker (xe2x80x9cbiomarkerxe2x80x9d) of the disease/progression of the disease (reduced abundance of transcripts or translation products will be highly indicative of the disease state). Similarly, a particular gene may be up-regulated (increased expression) in a particular disease. In such a disease, therefore, transcripts or translation products of the particular gene will be more abundant than the same transcripts or translation products in a non-diseased individual. Thus, since the abundance of particular transcripts or translation products may be ascertained using, for example, microarray technology, the particular gene (or rather its transcripts or translation products) will act as a marker (xe2x80x9cbiomarkerxe2x80x9d) of the disease/progression of the disease (increased abundance of transcripts or translation products will be highly indicative of the disease state).
The use of differential gene expression as biomarkers is described in, for example, Molecular Classification of Cancer: Class Discovery and Class Prediction by Gene Expression Monitoring. T. R. Golub, D. K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J. P. Mesirov, H. Coller, M. L. Loh, J. R. Downing, M. A. Caligiuri, C. D. Bloomfield, and E. S. Lander. Science, 286: 531-537 (1999), and Delineation of Prognostic Biomarkers in Prostate Cancer. Dhanasekaran S M, Barrette T R, Ghosh D, Shah R, Varambally S, Kurachi K, Pienta K J, Rubin M A, Chinnaiyan A M. Nature, 412: 822-825 (2001), both of which are incorporated herein by reference.
xe2x80x9cMicroarray technologyxe2x80x9d or xe2x80x9cDNA microarray technologyxe2x80x9d is now state of the art. The use of such technology in the analysis of differential gene expression is described in, for example, Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. R. Heller et al. Proc. Natl. Acad. Sci (USA), 94: 2150-2155 (1997), Quantitative Monitoring of Gene Expression Patterns with a Complimentary DNA Microarray. M. Schena et al., Science, 270: 467-470 (1995), and Expression monitoring by hybridisation to high-density oligonucleotide arrays. D. J. Lockhart et al., Nature Biotechnology, 14: 1675-1680 (1996), all of which are incorporated herein by reference.
A non-human mammal or an animal cell that is xe2x80x9cgenetically-modifiedxe2x80x9d is heterozygous or homozygous for a modification that is introduced into the non-human mammal or animal cell, or into a progenitor non-human mammal or animal cell, by genetic engineering. The standard methods of genetic engineering that are available for introducing the modification include homologous recombination, viral vector gene trapping, irradiation, chemical mutagenesis, and the transgenic expression of a nucleotide sequence encoding antisense RNA alone or in combination with catalytic ribozymes. Preferred methods for genetic modification are those which modify an endogenous gene by inserting a xe2x80x9cforeign nucleic acid sequencexe2x80x9d into the gene locus, e.g., by homologous recombination or viral vector gene trapping. A xe2x80x9cforeign nucleic acid sequencexe2x80x9d is an exogenous sequence that is non-naturally occurring in the gene. This insertion of foreign DNA can occur within any region of the PDE11A gene, e.g., in an enhancer, promoter, regulator region, noncoding region, coding region, intron, or exon. The most preferred method of genetic engineering is homologous recombination, in which the foreign nucleic acid sequence is inserted in a targeted manner either alone or in combination with a deletion of a portion of the endogenous gene sequence.
By a PDE11A gene that is xe2x80x9cfunctionally disruptedxe2x80x9d is meant a PDE11A gene that is genetically modified such that the cellular activity of the PDE11A polypeptide encoded by the disrupted gene is decreased in cells that normally express a wild type version of the PDE11A gene. When the genetic modification effectively eliminates all wild type copies of the PDE11A gene in a cell (e.g., the genetically-modified, non-human mammal or animal cell is homozygous for the PDE11A gene disruption or the only wild type copy of PDE11A gene originally present is now disrupted), then the genetic modification results in a reduction in PDE11A polypeptide activity as compared to an appropriate control cell that expresses the wild type PDE11A gene. This reduction in PDE11A polypeptide activity results from either reduced PDE11A gene expression (i.e., PDE11A mRNA levels are effectively reduced and produce reduced levels of PDE11A polypeptide) and/or because the disrupted PDE11A gene encodes a mutated polypeptide with reduced function or stability as compared to a wild type PDE11A polypeptide. Preferably, the activity of PDE11A polypeptide in the genetically-modified, non-human mammal or animal cell is reduced to 50% or less of wild type levels, more preferably, to 25% or less, and, even more preferably, to 10% or less of wild type levels. Most preferably, the PDE11A gene disruption results in non-detectable PDE11A activity.
By a xe2x80x9cgenetically-modified, non-human mammalxe2x80x9d containing a functionally disrupted PDE11A gene is meant a non-human mammal that is originally produced, for example, by creating a blastocyst or embryo carrying the desired genetic modification and then implanting the blastocyst or embryo in a foster mother for in utero development. The genetically-modified blastocyst or embryo can be made, in the case of mice, by implanting a genetically-modified embryonic stem (ES) cell into a mouse blastocyst or by aggregating ES cells with tetraploid embryos. Alternatively, various species of genetically-modified embryos can be obtained by nuclear transfer. In the case of nuclear transfer, the donor cell is a somatic cell or a pluripotent stem cell, and it is engineered to contain the desired genetic modification that functionally disrupts the PDE11A gene. The nucleus of this cell is then transferred into a fertilized or parthenogenetic oocyte that is enucleated; the embryo is reconstituted, and developed into a blastocyst. A genetically-modified blastocyst produced by either of the above methods is then implanted into a foster mother according to standard methods well known to those skilled in the art. A xe2x80x9cgenetically-modified, non-human mammalxe2x80x9d includes all progeny of the mammals created by the methods described above, provided that the progeny inherit at least one copy of the genetic modification that functionally disrupts the PDE11A gene. It is preferred that all somatic cells and germline cells of the genetically-modified mammal contain the modification. Preferred non-human animals that are genetically-modified to contain a disrupted PDE11A gene include rodents, such as mice and rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, and ferrets.
By a xe2x80x9cgenetically-modified animal cellxe2x80x9d containing a functionally disrupted PDE11A gene is meant an animal cell, including a human cell, created by genetic engineering to contain a functionally disrupted PDE11A gene, as well as daughter cells that inherit the disrupted PDE11A gene. These cells may be genetically-modified in culture according to any standard method known in the art. As an alternative to genetically modifying the cells in culture, non-human mammalian cells may also be isolated from a genetically-modified, non-human mammal that contains a PDE11A gene disruption. The animal cells of the invention may be obtained from primary cell or tissue preparations as well as culture-adapted, tumorigenic, or transformed cell lines. These cells and cell lines are derived, for example, from endothelial cells, epithelial cells, islets, neurons and other neural tissue-derived cells, mesothelial cells, osteocytes, lymphocytes, chondrocytes, hematopoietic cells, immune cells, cells of the major glands or organs (e.g., testicle, liver, lung, heart, stomach, pancreas, kidney, and skin), muscle cells (including cells from skeletal muscle, smooth muscle, and cardiac muscle), exocrine or endocrine cells, fibroblasts, and embryonic and other totipotent or pluripotent stem cells (e.g., ES cells, ES-like cells, and embryonic germline (EG) cells, and other stem cells, such as progenitor cells and tissue-derived stem cells). The preferred genetically-modified cells are ES cells, more preferably, mouse or rat ES cells, and, most preferably, human ES cells.
By an xe2x80x9cES cellxe2x80x9d or an xe2x80x9cES-like cellxe2x80x9d is meant a pluripotent stem cell derived from an embryo, from a primordial germ cell, or from a teratocarcinoma, that is capable of indefinite self renewal as well as differentiation into cell types that are representative of all three embryonic germ layers.
By a xe2x80x9cPDE11A genexe2x80x9d is meant the nucleic acid sequences encoding the polypeptides disclosed in Fawcett, 2000, (human PDE11A1, GenBank Accession No. AJ251509), WO 00/40733 (human PDE11A1 and PDE11A2), or Yuasa, 2000 (human PDE11A3 and PE11A4, GenBank Accession No. AB036704 and AB038041), as well as any human allelic variants and any mammalian sequences encoding homologues having PDE11A activity and their allelic variants. By a xe2x80x9cPDE11 polypeptidexe2x80x9d is meant the phosphodiesterases disclosed in Fawcett, 2000 or Yuasa, 2000, as well as any polypeptides encoded by any human allelic variants and any mammalian homologues having PDE11A activity. As used herein, the term xe2x80x9chomologuexe2x80x9d means a protein that is evolutionarily related to and shares substantial structural and functional similarity with a reference protein in a different species (e.g., human and mouse PDE11A polypeptides).
By xe2x80x9cPDE11A polypeptide activityxe2x80x9d or xe2x80x9cPDE11A polypeptide-like activityxe2x80x9d or xe2x80x9cPDE11A activityxe2x80x9d is meant the hydrolysis of cAMP or cGMP by a polypeptide encoded by a PDE11A gene. Such activity in a cell can be modulated at the level of PDE11A expression (e.g., by changing the amount of polypeptide that is effectively present within a cell) or by modifying the particular functional characteristics of each PDE11A polypeptide molecule (e.g., by changing the Km of hydrolysis for the mutated polypeptide).
By xe2x80x9cmodulatesxe2x80x9d is meant increases or decreases (including a complete elimination).
By an agent that is xe2x80x9cselectivexe2x80x9d for modulating PDE11A activity is meant an agent that primarily effects PDE11A while producing little effect on another PDE at the same concentration of agent. For example, if an agent is selective for inhibiting PDE11A, the IC50 of the agent for PDE11A is at least 20 fold, more preferably, at least 30 fold, even more preferably, at least 50 fold, and, even more preferably, at least 100 fold, lower in concentration as compared to the IC50 for one or more PDEs from the group of PDEs 1-10. Preferably, the agent is selective for PDE11A as compared to PDE7-10, and, even more preferably, as compared to all of PDEs 1-10, especially PDE3-6.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims. While the invention is described in connection with specific embodiments, it will be understood that other changes and modifications that may be practiced are also part of this invention and are also within the scope of the appendant claims. This application is intended to cover any equivalents, variations, uses, or adaptations of the invention that follow, in general, the principles of the invention, including departures from the present disclosure that come within known or customary practice within the art, and that are able to be ascertained without undue experimentation. Additional guidance with respect to making and using nucleic acids and polypeptides is found in standard textbooks of molecular biology, protein science, and immunology (see, e.g., Davis et al., Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York, N.Y., 1986; Hames et al., Nucleic Acid Hybridization, IL Press, 1985; Molecular Cloning, Sambrook et al., Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley and Sons; Current Protocols in Human Genetics, Eds. Dracopoli et al., John Wiley and Sons; Current Protocols in Protein Science, Eds. John E. Coligan et al., John Wiley and Sons; and Current Protocols in Immunology, Eds. John E. Coligan et al., John Wiley and Sons). All publications mentioned herein are incorporated by reference in their entireties.